-->PONE-D-25-50808-->-->PCR-Based Detection and Phylogenetic Analysis of Candidatus Liberibacter asiaticus in Citrus Orchards Across Nepal-->-->PLOS ONE

Dear Dr. Oliya,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Nishant Kumar, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Partly

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: I Don't Know

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The paper found satisfactory after my review. The author has done all the requisites related to the title mentioned. The results are highly correlated. Research work is highly appreciated. Data produced is genuine.

Reviewer #2: Lack of background depth: The study briefly mentions the global significance of citrus greening but does not explicitly state the current status or prior studies in Nepal. How this study builds upon or differs from existing regional work would strengthen its significance.

Limited sequencing depth: Only five samples were subjected to Sanger sequencing, which is insufficient for strong conclusions on genetic diversity.

No details on PCR validation: The abstract does not mention whether positive and negative controls, sensitivity checks, or replicates were used to confirm PCR reliability.

No environmental or management correlation: The study does not discuss about the factors like altitude, climate, or management practices that might influence disease spread.

The abstract could better link the molecular findings to practical outcomes for farmers or policy.

Future management or diagnostic development prospects are mentioned vaguely without specific pathways.

Time duration to reach the laboratory from collection sites and locations from where the samples have been collected are not mentioned.

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Reviewer #1: Yes:Dr. Kuljinder Kaur

Reviewer #2: No

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-->

-->PONE-D-25-50808R1-->-->PCR-Based Detection and Phylogenetic Analysis of Candidatus Liberibacter asiaticus in Citrus Orchards Across Nepal-->-->PLOS One

Dear Dr. Oliya,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Apr 11 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Nishant Kumar, Ph.D

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #2: Yes

Reviewer #3: Partly

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #2: Yes

Reviewer #3: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #2: (No Response)

Reviewer #3: Comments to the Author

The author addresses an important phytopathological problem (HLB) and provides country-wide molecular detection data from Nepal, which has regional significance. The dataset 1026 samples is strong, and the study attempts both diagnostics and phylogenetics. However, scientific depth, methodological rigor are insufficient for PLOS ONE in the current form. Several major conceptual and technical limitations must be addressed.

Major comments

Study primarily relies on conventional PCR and 16S rRNA sequencing, which are standard approaches. The claim of being a “comprehensive genetic characterization” is overstated given the limited depth of analysis.

Out of 1026 samples, only five were sequenced without justification of selection criteria. This is insufficient to represent genetic diversity across Nepal.

Phylogeny is based only on a short 16S rRNA fragment (~500 bp), which lacks resolution for intra-species variation. Use of Neighbor-Joining further weakens evolutionary interpretation.

“Candidatus” should be italicized, while “Liberibacter asiaticus” should not be italicized as currently presented. Please ensure consistent and correct formatting of this nomenclature throughout the manuscript.

References are not formatted according to the journal’s guidelines. Please revise all citations and the reference list to strictly comply with the required journal format.

Manuscript lacks essential tables. It is recommended to include tables for primer details, criteria for sample selection for sequencing, and a summary of accession numbers to improve clarity and organization.

Minor Comments

Line 24–25: “Ca. L. asiaticus (Ca. L. asiaticus)” → redundant repetition.

Line 39–40: Sentence is repetitive and can be shortened for clarity.

Line 58: “Gram-negative bacteria Candidatus Liberibacter spp.” → should be “Gram-negative bacterium” or restructure sentence.

Line 208: “25-well thermal cycler” → incorrect terminology; should specify PCR machine model.

Line 209–211: PCR reaction volumes do not sum correctly to 15 µl; needs correction.

Line 370: Missing space before citation “[41]”

Line 323–324: “citrus tresteza virus” → spelling error (tristeza).

Conclusion section: Repeats statements from abstract; should be more concise and focused on implications.

Please Cite these below references

Bharsakale, R. D., Alex, B. K., Gubyad, M. G., Kokane, A. D., Kokane, S. B., Shukla, P. K., & Ghosh, D. K. (2025). Development of a novel duplex PCR-lateral flow immunoassay (d-PCR-LFIA) for simultaneous detection of Candidatus Liberibacter asiaticus and citrus tristeza virus. Tropical Plant Pathology, 50(1), 77.

Ghosh, D., Kokane, S., Savita, B. K., Kumar, P., Sharma, A. K., Ozcan, A., ... & Santra, S. (2022). Huanglongbing pandemic: current challenges and emerging management strategies. Plants, 12(1), 160.

For the listed above comments, I would recommend to the editor accept with major revisions for this manuscript.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #2: No

Reviewer #3: Yes:Dilip Kumar Ghosh

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Attachments

Attachment

Submitted filename: Comments to the Author.docx

PCR-Based Detection and Phylogenetic Analysis of Candidatus Liberibacter asiaticus in Citrus Orchards Across Nepal

PONE-D-25-50808R2

Dear Dr. Oliya,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nishant Kumar, Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

-

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #3: Authors have addressed all the major and minor comments raised during the previous rounds of review. The manuscript has been substantially improved in terms of clarity, methodological transparency, and scientific accuracy. All previously identified concerns have been satisfactorily resolved, and the manuscript is now scientifically sound and suitable for publication.

Reviewer #4: The manuscript is well-structured, clearly written, and presents scientifically sound work. The objectives are clearly defined, and the methodology is appropriate and adequately described, allowing reproducibility. The data are presented in a coherent manner and strongly support the conclusions drawn by the authors. The statistical analyses are robust and properly interpreted. The discussion effectively contextualizes the findings within the existing literature, highlighting the significance and novelty of the work. Overall, this manuscript makes a valuable contribution to the field and is suitable for publication in its current form.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #3: No

Reviewer #4: No

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